Alkaline phosphatases (AP) are dimeric, zinc-containing, non-specific phosphomono-esterases which occur in prokaryotic and eukaryotic organisms e.g. in E. coli and mammals (McComb et al., 1979 Alkaline Phosphatases Plenum Press, New York). A comparison of the primary structure of various alkaline phosphatases showed that there is a high degree of homology (25-30% homology between E. coli and mammalian AP; Millàn, 1988 Anticancer Res. 8, 995-1004; Harris, 1989 Clin. Chim. Acta 186, 133-150).
In humans and higher animals the AP family consists of four members which are coded in different gene loci (Millàn, 1988 Anticancer Res. 8, 995-1004; Harris 1989 Clin. Chim. Acta 186, 133-150). The family of alkaline phosphatases includes the tissue-specific APs (placental AP (PLAP), germ cell AP (GCAP) and intestinal AP (IAP)) and the non-tissue-specific APs (TnAP) which are mainly located in the liver, kidney and bones.
An important property of the previously known APs is the large variability in the catalytic activity of mammalian APs which have a 10-100-fold higher kcats value than E. coli AP. Among the mammalian APs the APs from the bovine intestine (bIAP) exhibit the highest specific activities. This property makes the bIAPs attractive for biochemical applications such as e.g. the use of corresponding enzyme conjugates as a diagnostic reagent or for dephosphorylating DNA. The existence of various alkaline phosphatases from the bovine intestine which have different levels of specific activity is described in EP 0955 369 and Manes et al. (1998), J. Biol. Chem. 273 No. 36, 23353-23360. Up to now recombinant expression of eukaryotic alkaline phosphatases of low activity (up to 3000 U/mg) has been described in various eukaryotic cell lines such as CHO cells (bIAP I/WO 93/18139; Weissig et al. 1993, Biochem. J. 260, 503-508), COS cells (human placental AP/Berger et al. 1987 Biochemistry 84, 4885-4889) or baculovirus expression system (human placental AP/Davis et al. 1992, Biotechnology 10, 1148-1150). The expression of APs having a higher activity (spec. activity>3000 U/mg) from the bovine intestine in CHO cells has also been described (bIAP II, III and IV/Manes et al. 1998, J. Biol. Chem. 273 No. 36, 23353-23360). However, a disadvantage of expressing alkaline phosphatases in these expression systems is the low expression rate which makes it uneconomical to produce eukaryotic alkaline phosphatase recombinantly.
Although in principle it is possible to express eukaryotic alkaline phosphatases in prokaryotic expression hosts such as E. coli (human placental AP/Beck and Burtscher, 1994 Protein Expression and Purification 5, 192-197), the alkaline phosphatases expressed in prokaryotes have no glycosylation which is essential especially for the preparation of enzyme conjugates depending on the conjugate method.
Alkaline phosphatase is often used as an enzyme conjugate in the form of a complex with an antibody. In this case the alkaline phosphatase is conjugated with an antibody which is directed against a particular antigen. This antigen is firstly bound in a first reaction by an antibody immobilized on a vessel wall which recognizes a different epitope on the target antigen than does the antibody-AP conjugate. This antibody-antigen complex is then detected in a second reaction by the binding of the antibody-AP conjugate. False-positive results occur repeatedly in such tests and are caused by an unspecific binding of the antibody-AP conjugate to the vessel wall or to the first antibody. These interferences can be prevented by adding an excess of a conjugate containing an inactive or weakly active AP mutant as an interference-eliminating protein. However, in order to act very specifically as an interference-eliminating protein, the AP mutant must, in addition to having a low activity or no activity, also have essentially the same tertiary and quaternary structure.
Hence the object of the present invention is to use directed mutagenesis to produce mutants of alkaline phosphatase as an interference-eliminating protein which are only very weakly-active or completely inactive but whose amino acid sequence is only slightly modified and have a tertiary and quaternary structure that is changed as little as possible. Another object of the invention is to develop a robust and stable expression method for producing glycosylated eukaryotic alkaline phosphatase mutants which also enables an economical production of a corresponding alkaline phosphatase mutant due to the high expression rate.